... CHARLES$, B. T. DRISCOLL#, B. PRITHIVIRAJ". D. L. SMITH"*. "Plant Science Department and #Department of Natural Resource Science, Macdonald ...
Journal of Agricultural Science (2002), 138, 293–300. # 2002 Cambridge University Press DOI : 10.1017\S0021859602001995 Printed in the United Kingdom
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Bradyrhizobium japonicum mutants allowing improved nodulation and nitrogen fixation of field-grown soybean in a short season area H. Z H A N G", F. D A O U S T#, T. C. C H A R L E S$, B. T. D R I S C O L L#, B. P R I T H I V I R A J" D. L. S M I T H"* " Plant Science Department and # Department of Natural Resource Science, Macdonald Campus of McGill University, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec, Canada H9X 3V9 $ Department of Biology, University of Waterloo, Ontario, Canada N2L 3G1 (Revised MS received 14 November 2001)
SUMMARY In short-season soybean production areas, low soil temperature is potentially a major factor limiting plant growth and yield. Although Bradyrhizobium japonicum strain 532 C is widely used in inoculants in Canadian soybean production, and USDA 110 is widely used in American soybean production, they are both inhibited by low temperatures. Genistein is an important plant-to-bacteria signal compound secreted by soybean roots. The addition of genistein has proven to be an effective means of generating increases in nitrogen fixation and yield but genistein is expensive. We used UV mutagenesis to make 10 mutants from USDA 110 that express nod genes without the addition of plant-to-bacteria signal compounds such as genistein. A field experiment was conducted at the Lods Agronomy Research Centre in southwestern Quebec in 1998 and 1999. The treatments consisted of factorial combinations of inoculant type (no inoculant (control) and inoculants containing the mutants Bj 30050, Bj 30051, Bj 30052, Bj 30053, Bj 30054, Bj 30055, Bj 30056, Bj 30057, Bj 30058, Bj 30059 and the wild type USDA110 or 532 C) and soybean cultivar (Bayfield and Maple Glen). The experiment was structured following a randomized complete block design with four blocks. Data were collected on nodule number, nodule dry weight, shoot nitrogen yield and total nitrogen fixation at five development stages. Averaged over the 2 years of the study, when pods were 2 cm long at one of the four uppermost nodes on the main stem (R4), inoculation with Bj 30055 and Bj 30058 resulted in greater nodule number (23 and 14 %, respectively), nodule dry weight (16 and 13 %, respectively), shoot nitrogen yield (19 and 21 %, respectively) and total nitrogen fixation (10n9 and 12n7 %, respectively) than 532 C, which is currently used in Canadian inoculants. The cultivar Bayfield produced more nodules, and higher nodule weight than Maple Glen, but there were no differences between the cultivars for shoot nitrogen yield and total nitrogen fixation. INTRODUCTION The formation of effective soybean nodules is a complex and highly regulated process that requires production and exchange of specific molecular signals between the host plant and the bacterial symbiont. The most effective of the plant-to-bacteria signal molecules is the isoflavone genistein (Kosslak et al. 1987), which is secreted by soybean roots. Genistein induces B. japonicum nodulation (nod ) genes, by binding the NodD1 or NodV proteins. The products of the nod genes are Nod factors, which have been * To whom all correspondence should be addressed. Email : dsmith!macdonald.mcgill.ca
identified as lipo-chito-oligosaccharides (Long 1996). Nod factors are responsible for many of the early stages of nodule development (Sanjuan et al. 1992). Soybean is a subtropical legume that requires root zone temperatures (RZTs) in the range of 25 to 30 mC for optimal symbiotic activity. At lower temperatures, the expression of the nod genes is suboptimal (Zhang et al. 1996 b), resulting in a delay in the onset of nodulation (Zhang & Smith 1996 b). In regions such as Canada, low soil temperature is considered to be the major factor potentially limiting soybean growth and symbiotic nitrogen fixation (Whigham & Minor 1978). All stages of symbiotic establishment investigated to date (root hair curling, infection thread formation and penetration, nodule development and
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function) are inhibited by suboptimal temperatures (less than 25 mC) (Lindemann & Ham 1979). The addition of genistein has proven to be an effective means of generating increases in nitrogen fixation and yield (Zhang & Smith 1996 b). Zhang & Smith (1996 b) reported that two strains of B. japonicum responded differently to signal-molecule additions. Genistein is an expensive chemical, and therefore it would be beneficial to find a different solution to the problem of soybean nodulation at low root-zone temperatures. Our research group used UV mutation and made 10 mutants from USDA 110 in which expression of nod genes occurs independently of genistein (unpublished data). However, until now the effects of these mutants on nodulation, plant development and nitrogen fixation under field conditions in a short-season, cool-spring soil environment have not been reported. Therefore, in this work we have tested the following hypotheses : (1) there is variability among B. japonicum mutants for ability to nodulate soybean plants and increase soybean nitrogen fixation under field conditions in a cool-spring short-season area ; (2) B. japonicum mutants that can express nod genes without exposure to plant-to-bacteria signal compounds will be able to overcome low RZT inhibition of soybean nodulation, leading to improved soybean development under field conditions in a coolspring short-season area. Hence, the goal of this study was to evaluate mutants that express nod genes without exposure to plant-to-bacteria signals for ability to improve soybean nodulation and N fixation # in short-season areas with low spring soil temperatures. MATERIALS AND METHODS Isolation of a genistein-independent B. japonicum strain The wild type B. japonicum strain USDA 110 was subjected to UV mutagenesis (30 000 µJ) to generate potential genistein-independent mutants. A reporter plasmid (pZB32, obtained from G. Stacey, University of Tennessee, Knoxville, Tennessee), consisting of a nodY : : lacZ gene fusion, was then introduced into the mutagenized cells by electroporation (Hattermann & Stacey 1990). Tetracycline-resistant transformants were selected on YEM plates containing X-Gal (80 µg\ml), but no genistein. Blue colonies were considered to be putative mutants. To confirm the stability of the putative constitutive mutants isolated, 10 candidate strains were cured of the reporter plasmid. The plasmid was then reintroduced by conjugation with E. coli S 17.1 carrying pZB32, and the phenotype was verified on YEM and TY plates with tetracycline and X-Gal. All putative mutants exhibited blue colonies under such conditions, confirming the mutation.
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Bacterial culture The field experiment used the mutants Bj 30050, Bj 30051, Bj 30052, Bj 30053, Bj 30054, Bj 30055, Bj 30056, Bj 30057, Bj 30058 and Bj 30059, and the wild types 532 C and USDA 110 as inoculants to test whether inoculation with the mutants would result in nodulation and nitrogen fixation levels that were greater than 532 C and USDA 110. The inocula were produced by culturing the above 10 mutants and 2 strains in YEM broth (Vincent 1970) in 250 ml flasks shaken at 125 rpm at 25 mC. When the subcultures reached mid log phase, pure medium was used to dilute the inocula to an OD of 0n08 (equivalent to '#! 10) cell\ml) (Bhuvaneswari et al. 1980). Site preparation and field layout Each plot was 4n5i3n2 m and consisted of eight rows of plants, with 0n4 m between rows. The space between adjacent blocks was 1 m. The space between plots was 0n8 m. Available soil nitrogen was estimated by the inclusion of one plot of a non-nodulating Evans line of soybean, randomly located, in each block. The experiments were carried out at the Lods Agronomy Research Center, McGill University, Macdonald Campus. The soil was a Chicot light sandy loam (mixed, frigid Typic Hapludalf) in 1998 and 1999. Oat and barley were planted in 1997 and corn was planted in 1998 at the 1998 and 1999 sites, respectively. In each case the stems and leaves were incorporated into the soil after harvest through fall ploughing. In both years, potassium and phosphate were provided by a spring application of muriate of potash (110 kg\ha of 0-0-60) and triple superphosphate (220 kg\ha of 0-460) following the recommendations of a soil test. Planting method Seeds of the soybean cultivars Maple Glen and Bayfield and a non-nodulating Evans line were surface-sterilized in sodium hypochlorite (2 % solution), then rinsed several times with distilled water. These seeds were planted on 15 May in 1998 and 18 May in 1999. Twenty ml of mutant\strain culture per 1 m of row were applied evenly by syringe directly onto the seed along the furrow. Alcohol sterilization of the implements was used to prevent crosscontamination throughout planting and all subsequent data collection procedures. Following emergence, seedlings reached a stand of 500 000 plants\ha (20 plants\m of row, with an average inter-plant distance of 5 cm within the row and 40 cm between rows). Where initial stands were greater than 500 000 plants\ha the plots were hand thinned at the seedling stage. Experimental design The field experiment was a 13i2 factorial organized
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Fig. 1. The average daily air temperature - -#- -, soil temperature (at a depth of 5 cm ; –$–) and precipitation –X– during the 1998 soybean growing season (Ste-Anne-de-Bellevue, Quebec, Canada).
in a randomized complete block design with four blocks. The treatments consisted of factorial combinations of 13 inocula (strains 532 C and USDA 110 ; 10 mutants, our designations, Bj 30050, Bj 30051, Bj 30052, Bj 30053, Bj 30054, Bj 30055, Bj 30056, Bj 30057, Bj 30058, Bj 30059, an uninoculated control), with two soybean cultivars (Maple Glen and Bayfield). Strain 532 C was included as it is widely used in Canadian soybean inoculants. Strain USDA 110 was included as it is widely used in inoculants in the USA and the mutants are derived from this strain. We chose USDA 110 for the production of the mutants as its genetics have been well characterized. Harvest and data collection Daily average air temperature, average soil temperatures at a depth of 5 cm and precipitation were recorded at the Macdonald Campus weather station. The classification of soybean growth stages followed those of Fehr et al. (1971). Plant samples (10 plants in each) were harvested from each plot at four development stages : (1) V3, three nodes on the main stem with fully developed leaves beginning with the unifoliate nodes (20 June in 1998 and 24 June in 1999) ; (2) R1, one open flower at any node on the main stem (28 June in 1998 and 2 July in 1999) ; (3) R4, pods 2 cm long at one of the four uppermost nodes on the main stem with a fully developed leaf (28 July in 1998 and 4 August in 1999) ; (4) R8, 95 % of the pods at their mature pod colour (14 August in 1998 and 18 August in 1999). The final plants were harvested with a small plot combine (Wintersteiger,
Salt Lake City, UT) at harvest maturity, oven-dried at 70 mC for at least 48 h and weighed. Data were collected on nodule number, nodule dry weight, shoot nitrogen yield and total nitrogen fixed. The nitrogen contents of the roots, stems (including leaves) and seeds were determined by Kjeldahl analysis (Kjeltec system, Tecator AB, Hoganas, Sweden). Shoot nitrogen yield was determined by adding stem and seed nitrogen contents together. The protein concentration was calculated by multiplying nitrogen concentration by 6n25. Total nitrogen fixed in roots, shoots, leaves and seeds was measured at the final harvest (after R8) and was calculated by the difference method, whereby the total N in the plot of non-fixing soybean plants was subtracted from the total N in each of the other plots, for each block. Statistical analysis Results were analysed statistically by analysis of variance using the Statistical Analysis System (SAS) computer package (SAS Institute Inc. 1990). When analysis of variance showed significant treatment effects, the least significant difference (LSD) test was applied to make comparisons among the means at the 0n05 level of significance (Steel & Torrie 1980). When differences occurred at levels of significance between 0n05 and 0n1 this is noted in the text. RESULTS Inoculation with Bj 30050, Bj 30051, Bj 30052, Bj 30053, Bj 30054, Bj 30056, Bj 30057 and Bj 30059
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Fig. 3. The effects of inoculated mutants and strains on soybean (Maple Glen and Bayfield) nodule number per plant in 1998 (A) and 1999 (B). Each value is plotted as the meanj.. (n l 10). The plant development stage at each harvest : (1) three nodes on the main stem with fully developed leaves beginning with unifoliate nodes (V3) ; (2) one open flower at any node on the main stem (R1) ; (3) pods 2 cm long at one of the four uppermost nodes on the main stem with a fully developed leaf (R4) ; (4) 95 % of the pods having reached their mature pod colour (R8). Key : –$–, Bj 30055 ; –#–, Bj 30058 ; –X–, 532 C ; –W–, USDA 110 ; – –, Control.
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Table 1. Main effects of Bradyrhizobium japonicum strains\mutants and soybean cultivar on soybean nodule number per plant, nodule weight per plant, and shoot nitrogen yield (kg\ha) at harvest 3 (R4) in 1998 and in 1999* Nodule number\ plant Treatment Inoculant 532 C Bj 30055 Bj 30058 USDA 110 Uninoculated .. (.. l 77) Cultivar Maple Glen Bayfield .. (.. l 77)
Nodule weight (mg\plant)
Shoot nitrogen yield (kg\ha)
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1999
89n1 110n5 103n9 80n7 70n8 4n0
87n0 106n3 97n3 80n8 72n6 3n6
436n0 505n5 491n8 379n4 225n3 22n9
427n0 497n0 483n0 364n3 224n7 14n4
300 347 357 284 218 16
301 366 366 283 227 18
85n0 96n9 4n0
82n1 95n4 5n0
391n5 423n6 3n6
385n4 413n0 1n4
294 309 6
308 309 3
* Mean nodule number per plant, nodule dry weight and shoot nitrogen yield were derived from 10 plants from each plot at R4.
Nodule dry weight (mg)/plant
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Fig. 4. The effects of inoculated mutants and strains on soybean (Maple Glen and Bayfield) nodule dry weight per plant in 1998 (A) and 1999 (B). Each value is plotted as the meanj.. (n l 10). The plant development stage at each harvest : (1) three nodes on the main stem with fully developed leaves beginning with unifoliate nodes (V3) ; (2) one open flower at any node on the main stem (R1) ; (3) pods 2 cm long at one of the four uppermost nodes on the main stem with a fully developed leaf (R4) ; (4) 95 % of the pods having reached their mature pod colour (R8). Key : –$–, Bj 30055 ; –#–, Bj 30058 ; –X–, 532 C ; –W–, USDA 110 ; – –, Control.
resulted in values of measured variables that were not different from the uninoculated control. Because of this the remainder of the results section will focus largely on results from Bj30055, Bj30058, USDA 110 and 532 C.
Effects of strains and mutants on nodulation and nodule mass The seasonal soil temperature data in 1998 and 1999 showed that the average daily RZTs at a depth of
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Fig. 5. The effects of inoculated mutants and strains on soybean (Maple Glen and Bayfield) shoot nitrogen yield in 1998 (A) and 1999 (B). Each value is plotted as the meanj.. (n l 10). The plant development stage at each harvest : (1) three nodes on the main stem with fully developed leaves beginning with unifoliate nodes (V3) ; (2) one open flower at any node on the main stem (R1) ; (3) pods 2 cm long at one of the four uppermost nodes on the main stem with a fully developed leaf (R4) ; (4) 95 % of the pods having reached their mature pod colour (R8). Key : –$–, Bj 30055 ; –#–, Bj 30058 ; –X–, 532 C ; –W–, USDA 110 ; – –, Control.
5 cm were below 25 mC for most of the time (Figs 1 and 2). The air temperature data in 1998 were higher than those in 1999. Thus, low soil temperature was potentially a limiting factor to soybean nodulation in our study. Each strain or mutant had a different effect on nodulation and shoot nitrogen yield at each harvest (Figs 3–5). At R4 all strains and mutants had the highest values for nodule number, nodule weight and shoot nitrogen yield, thus, while all the data are shown in the graphs, we will describe the effects at R4 as examples (Table 1). Compared with the uninoculated control, inoculation with these strains and mutants increased nodule number and nodule weight. At R4, compared to 532 C, Bj 30055 and Bj 30058 inoculation increased nodule number by 24 % and 17 % in 1998, respectively, and 23 % and 12 %, respectively, in 1999. Compared to USDA 110 they increased nodule number by 37 % and 29 %, respectively, in 1998, and 32 % and 20 %, respectively, in 1999 (Table 1). Bayfield produced more nodules than Maple Glen. Similarly, inoculation with the mutants enhanced the nodule dry weight ; inoculation with Bj 30055 and Bj 30058 resulted in nodule dry weights of 505n5 mg\plant and 491n8 mg\plant as compared to 436 mg\plant for 532 C or 375n4 mg\plant for
USDA 110 during the 1998 season. Similar increases in nodule dry weight were observed in the 1999 season (Figs 3 & 4). Strain and mutant effects on shoot nitrogen yield and total nitrogen fixation At R4, compared to USDA 110, inoculation with Bj 30055 and Bj 30058 increased shoot nitrogen accumulation by 22 and 26 %, respectively, in 1998, and by 29 % in both in 1999. Compared with 532 C, inoculation by Bj30055 and Bj30058 increased shoot nitrogen accumulation by 16 and 19 %, respectively, in 1998, and by 22 % in both in 1999. Shoot nitrogen yield reached the highest level at R4. There was no difference in shoot nitrogen yield between the two cultivars (Table 1). Total nitrogen fixation due to inoculation with Bj 30055 and Bj 30058 was greater than 532 C (9 and 10 %, respectively, in 1998 ; 13 and 16 %, respectively, in 1999). The total nitrogen fixation due to inoculation with Bj 30055 and Bj 30058 was greater than USDA 110 (14 and 15 %, respectively, in 1998 ; 11 and 14 %, respectively, in 1999) (Table 2). The mutant strains Bj 30055 and Bj 30058 enhanced total grain yield and grain protein production as
Nodulation and N fixation by Bradyrhizobium japonicum Table 2. Main effects of Bradyrhizobium japonicum strains\mutants and soybean cultivar on soybean total fixed N (kg\ha) at the final harvest in 1998 and 1999* Total fixed N (kg\ha) Treatment Inoculant 532 C Bj 30055 Bj 30058 USDA 110 Uninoculated .. (.. l 77) Cultivar Bayfield Maple Glen .. (.. l 77)
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148n5 161n4 162n9 141n8 123n6 3n6
141n3 159n6 163n4 143n4 126n7 3n1
158n1 149n8 4n5
151n7 140n4 5n1
* Values were derived from 10 plants from each plot collected at the final harvest.
compared with strains 532 C and USDA 110 ; inoculation with mutant Bj 30055 resulted in a seed yield of 1n92 t\ha and Bj 30058 produced 1n96 tonnes\ha as compared to 1n83 tonnes\ha by 532 C or 1n77 t\ha by USDA 110 during the 1998 season. Similarly, in the 1999 season inoculation with mutant Bj 30055 produced 1n89 t\ha and inoculation with Bj 30058 produced 1n91 t\ha, as compared with 1n76 t\ha and 1n68 t\ha with 532 C and USDA 110, respectively. These increases were all significant at P l 0n05. DISCUSSION We found that inoculation with 8 of the 10 mutants resulted in nodule number, nodule weight, shoot nitrogen yield and total nitrogen fixation values that were not different from the uninoculated control treatment. Our laboratory work (unpublished data) showed that these eight mutants did not produce detectable Nod factor (although, these mutants expressed the nod gene in the absence of genistein) independently of genistein but, under low temperature conditions, they produced more Nod factor than 532 C and USDA 110. It also showed that all mutants could induce nodule formation on soybean roots at room temperature. We postulated that the change(s) in gene structure of these mutants allowed them to produce more Nod factor, however, it may also have been the case that other unknown changes in gene structure made them ineffective under field conditions. Perhaps they were unable to survive the much more demanding conditions of the field environment, or compete effectively with indigenous B. japonicum strains. The low temperature limit of the nitrogen-fixing symbiosis is largely due to sensitivity on the part of
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the host plant but can be modified by the strain of bacterial symbiont used (Lie 1974). At present, existing soybean cultivars used in North America are derived from a relatively narrow genetic pool (Keyser & Cregan 1984) and no obvious relatives of soybean may be used to introduce cold tolerance (Sprent 1979). Thus, it is essential to look for effective strains or mutants that can improve soybean nitrogen fixation. Strain 532 C is widely used in Canadian soybean production, and USDA 110 is widely used in the United States, however, symbiotic N fixation by # both is inhibited by low temperature (Zhang & Smith 1996 b). All 10 mutants used in this work were derived from USDA 110. Our work has shown that the nodule dry weight produced due to inoculation with USDA 110 was less than following inoculation with 532 C, but nodule number, shoot nitrogen yield and total nitrogen fixation were not different between USDA 110 and 532 C. Genistein is an important signal compound secreted by soybean roots and it can induce nod gene expression in strains of B. japonicum. However, at lower temperatures, higher genistein concentrations were required for maximum nod gene expression (Zhang et al. 1996). Thus, the efficiency of genistein in terms of nod gene expression is lower at lower temperatures. At the same time, genistein concentration in soybean roots is low (Zhang & Smith 1996 a) at lower temperatures. Therefore, genistein can be a limiting factor in the development of the soybean nitrogenfixing symbiosis at low temperatures. Although adding genistein can improve soybean nodulation under low temperatures, it is expensive. Our research group selected for mutants (unpublished data) that can express nod genes without the addition of plant-to-bacteria signal compounds such as genistein in order to replace 532 C or reduce the inhibition of nitrogen fixation by low temperature. We found that inoculation with mutants Bj 30055 and Bj 30058 and strains 532 C and USDA 110 increased nodule number, nodule weight, shoot nitrogen yield and total nitrogen fixation in the field. Zhang & Smith (1994) reported that low temperature delayed the nodulation and Sprent (1979) speculated that, because the initial rise in nitrogen fixation was exponential, an increase of 10 % in the period of nodule activity of a grain legume could double the seasonal level of nitrogen fixed. Our results (unpublished data) indicated that the mutants Bj 30055 and Bj 30058 produced more Nod factor under low temperature than 532 C and USDA 110, although they did not produce it independently of genistein. Thus, mutants Bj 30055 and Bj 30058 were able to at least partially overcome the inhibition of nitrogen fixation by low temperature under field conditions. The only differences between cultivars were for nodule number and nodule dry weight, demonstrating a lack of any cultivar specificity for the strain\mutant
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effects. Nodulation of the uninoculated control demonstrated that indigenous B. japonicum strains existed in the field. In summary, this is the first report indicating the potential use of mutant strains of B. japonicum for soybean cultivation under cool season conditions. It is plausible that this might be due to the ability of the
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mutants to express nod genes in the absence of any plant-to-bacteria signal molecule, such as genistein, and this ability would have enabled them to overcome the low soil temperature inhibition of inter-organismal signalling. However, further studies are required to characterize these mutants before they can be used in commercial inoculants.
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Proceedings of the National Academy of Science, USA 89, 8789–8793. SAS (1990). SAS\STAT User’s guide. Version 6, Fourth Edition. Cary, USA : Statistical Analysis System Institute Inc. S, J. I. (1979). The Biology of Nitrogen Fixing Organisms. New York : McGraw-Hill. S, R. G. D. & T, J. H. (1980). Principles and Procedures of Statistics : A Biometrical Approach. New York : McGraw-Hill. V, J. M. (1970). A Manual for the Practical Study of Root Nodule Bacteria. Oxford : Blackwell Scientific. W, D. K. & M, H. C. (1978). Agronomic characteristics and environmental stress. In Soybean Physiology, Agronomy, and Utilization (Ed. G. A. Northman), pp. 77–118. New York : Academic Press. Z, F., C, T. C., P, B. & S, D. L. (1996). Inhibition of the expression of B. japonicum nod genes at low temperatures. Soil Biology and Biochemistry 28, 1579–1583. Z, F. & S, D. L. (1994). Effects of low-root zone temperature on the early stages of symbiosis establishment between soybean (Glycine max. (L.) Merr.) and Bradyrhizobium japonicum. Journal of Experimental Botany 279, 1467–1473. Z, F. & S, D. L. (1996a). Inoculation of soybean (Glycine max (L.) Merr.) with genistein-preincubated Bradyrhizobium japonicum or genistein directly into soil increases soybean protein and dry matter yield under short season conditions. Plant and Soil 179, 233–241. Z, F. & S, D. L. (1996b). Genistein accumulation in soybean (Glycine max (L.) Merr.) root systems under suboptimal root zone temperatures. Journal of Experimental Botany 47, 785–792.
